CREC, A NEW TOOL FOR METABOLIC MANIPULATIONS IN Escherichia coli.

GODOY MANUEL SANTIAGO; PETTINARI, MARIA JULIA

Córdoba

Congreso; SAMIGE; 2015

SAMIGE

Succinic acid is used as a specialty chemical in the agricultural, food, and pharmaceutical industries. Although succinic acid is currently produced from petroleum derived maleic anhydride, considerable interest in the fermentative production of succinate from sugars has emerged. Many genetic strategies have been performed to enhance succinate production in Escherichia coli, a natural but poor producer of this compound. The pleiotropic regulation over celullar functions carried out by two-component systems (TCSs) makes them interesting targets for genetic manipulations to achieve this purpose.CreC is a TCS responsive to the carbon source present in the media, whose absence was seen to enhance succinate production. In this work, additional strategies were tested to increase its production in creC mutants. Two plasmids carrying carboxylating enzymes were introduced in the parental and creC strains: pEcPpc, that overexpresses the carboxylating enzyme phosphoenolpiruvate caboxilase (Ppc) from E. coli, and pSBF2, that overexpresses the formate dehydrogenase from Candida boidini (Fdh). In both plasmids the genes are under the control of the lacZ promoter, and can be induced by IPTG.Two different concentrations of IPTG (0,1mM and 1M) were used to get a better estimation of the relative weight of the conversion catalyzed by Ppc and Fdh on succinate production. With the lowest concentration of IPTG, the creC mutant produced 3 times more succinate than the parental strain. These concentrations were only slightly higher than those observed in the absence of plasmids for both strains. However, when IPTG was supplied in a higher dose (1 mM), succinate production was triggered, with marked increases in all cases. The mutant strain overexpressing both plasmids produced more than four times what it had produced with 0,1 mM IPTG, and 40% more than the wild type in the same condition.In order to eliminate side products and increase NADH availability, the ethanol pathway was deleted and ackA was also eliminated to conserve carbon atoms in the form of acetyl-CoA, a substrate for succinate formation via the glyoxylate pathway. The double mutant creC, adhE and the triple mutant creC, adhE, ackA were cotransformed with pEcPpc and pSBF2, and succinate was measured in cultures of these strains grown in the same conditions previously described (NaHCO3 100mM, IPTG 0.1mM and 1mM). In contrast to what was expected, these strains did not present significantly higher amounts of succinate when compared to the simple mutant ∆creC harboring both plasmids. In all cases, a very marked increase was observed with higher amounts of IPTG, indicating that in the creC background, overexpression of Ppc and Fdh had an important effect on succinate production, while the mutations in adhE and ackA did not.These results indicate that CreC appears as a good candidate for genetic manipulation in order to improve a reduced compound of commercial interest, such as succinate.